sittingdrop

Sitting drop, also known as the sitting-drop vapor diffusion method, is a common crystallization technique used to grow crystals of biomolecules, especially proteins, for X-ray crystallography. It is a variant of the vapor diffusion approach in which the drop sits on a pedestal inside a sealed plate rather than hanging from a cover slip.

In the method, a small volume (often 0.2–2 μL) of protein solution mixed with precipitating agent is placed on a pedestal within a well that contains a larger reservoir of precipitant solution. Water vapor diffuses between the drop and the reservoir, gradually equalizing vapor pressure. This exchange alters the drop’s composition, driving changes in supersaturation that can lead to nucleation and crystal growth. The process is typically performed at a controlled temperature.

Sitting drops are widely used in automated and high-throughput crystallization screens because they are easy to load, monitor, and scale within multiwell plates. The approach is compatible with robotic handling, standardized plate formats, and integrated temperature control, making it a favored option in structural biology laboratories. Compared with hanging drops, sitting drops offer practical advantages for automation and throughput in many workflows.

Limitations include the fact that many proteins do not crystallize under initial screening conditions and require extensive optimization. Crystallization outcomes can be highly sensitive to small changes in condition, drop size, and evaporation; careful sealing and experimental design are important. Nevertheless, sitting-drop and hanging-drop vapor diffusion remain core methods for macromolecular crystallization.

See also: hanging drop, vapor diffusion, crystallization.